System and method for determining known DNA variants with temperature gradient electrophoresis

ABSTRACT

The present invention relates to a method of determining the genotype of a sample polynucleotide having at least a first variant site. At least a portion of the sample polynucleotide is amplified to obtain first amplicons, the first amplicons including the first variant site. The first amplicons are combined with first and second different polynucleotide controls, the first and second polynucleotide controls differing by at least one base therealong, the position of the at least one differing base corresponding to the first variant site of the sample polynucleotide. A plurality of first duplexes are prepared, each of at least some of the first duplexes comprising (i) a polynucleotide strand of one of the first amplicons and (ii) a complementary polynucleotide strand of the first polynucleotide control. A plurality of second duplexes are prepared, each of at least some of the second duplexes comprising (i) a polynucleotide strand of one of the first amplicons and (ii) a complementary polynucleotide strand of the second polynucleotide control. The first and second duplexes are subjected to temperature gradient electrophoresis (TGE) to obtain first and second electrophoresis data. The genotype of the first variant site of the sample polynucleotide is determiend based on the first and second electrophoresis data.

RELATED APPLICATIONS

[0001] The present application claims priority to U.S. ProvisionalApplication Nos. 60/395,614, filed Jul. 15, 2002 and 60/386,006, filedJul. 16, 2002. The present application is also a continuation of U.S.application Ser. No. 10/287,826, filed Nov. 5, 2002, which claimspriority to international application no. PCT/US01/274401, filed Sep. 4,2001, which claims priority to U.S. Provisional Application No.60/229,302, filed Sep. 1, 2000. Each of the foregoing applications isincorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to a system and method fordetermining variants in polynucleotides, such as DNA and in particulargenomic DNA.

BACKGROUND OF THE INVENTION

[0003] Efficient, fast and cost-effective techniques are still requiredfor analyses of single nucleotide polymorphisms (SNPs) and knownmutations associated with disease. Many methods have been developed forSNP/mutation genotyping (Landergren et al. 1998). The DNA-chip methodbased on hybridization may allow processing large numbers of samples,but it requires careful calibration of the signal when interpreting data(Wang et al. 1998). Single base extension (SBE) followed by separationwith capillary electrophoresis using automated sequencing instrument islimited by the length of the extension primer that can be synthesized bypresent technology. For instance, no more than 10 SBE products areseparated in a single capillary using the Applied Biosystems' SNaPshotkit (from Protocol of ABI Prism SNaPshot Multiplex Kit, 2000). Detectionof SBE reactions by mass spectrometry requires highly purified products,which can be costly and labor-intensive (Ross et al. 1998). For a morethorough review of the field, please see a number of papers published ina supplement to BioTechniques, June 2002, under the title—SNPs:Discovery of markers for disease.

SUMMARY OF THE INVENTION

[0004] A first aspect of the invention relates to a method ofdetermining the genotype of a sample polynucleotide having at least afirst variant site. The method may comprise amplifying at least aportion of the sample polynucleotide to obtain first amplicons, thefirst amplicons including the first variant site. The position of thefirst variant site along the sample nucleotide is preferably known. Thefirst amplicons may be combined with first and second differentpolynucleotide controls. The first and second polynucleotide controlsmay differ by at least one base therealong, the position of the at leastone differing base preferably corresponds to the position of the firstvariant site along the sample polynucleotide.

[0005] A plurality of first duplexes may be prepared. At least some andpreferably each of at least some of the first duplexes may comprise (i)a polynucleotide strand of one of the first amplicons and (ii) acomplementary polynucleotide strand of the first polynucleotide control.A plurality of second duplexes may be prepared. At least some andpreferably each of at least some of the second duplexes comprising (i) apolynucleotide strand of one of the first amplicons and (ii) acomplementary polynucleotide strand of the second polynucleotidecontrol.;

[0006] The first and second duplexes may be subjected to temperaturegradient electrophoresis (TGE) to obtain first and secondelectrophoresis data, which are indicative of the genotype of the firstvariant site. The genotype of the first variant site of the samplepolynucleotide may be determined based on the first and secondelectrophoresis data. The determination of the genotype may comprisedetermining a number of peaks present in the first electrophoresis dataand a number of peaks present in the second electrophoresis data.

[0007] The first duplexes and second duplexes may be subjected to TGEalong first and second different separation lanes, such as along thebores of different capillaries.

[0008] The first and second polynucleotide controls may be wild-typepolynucleotides.

[0009] The method may comprise amplifying at least a second differentportion of the sample polynucleotide to obtain second amplicons, thesecond amplicons including a second variant site of the samplepolynucleotide. The position of the second variant site along the samplepolynucleotide is preferably known. The second amplicons may be combinedwith third and fourth different polynucleotide controls, the third andfourth polynucleotide controls differing by at least one basetherealong. The position of the at least one differing base maycorrespond to the second variant site of the sample polynucleotide.

[0010] A plurality of third duplexes may be prepared. Each of at leastsome of the third duplexes may comprise (i) a polynucleotide strand ofone of the second amplicons and (ii) a complementary polynucleotidestrand of the third polynucleotide control. A plurality of fourthduplexes may be prepared. Each of at least some of the fourth duplexesmay comprise (i) a polynucleotide strand of one of the second ampliconsand (ii) a complementary polynucleotide strand of the fourthpolynucleotide control.

[0011] The third and fourth duplexes may be subjected to temperaturegradient electrophoresis (TGE) to obtain third and fourthelectrophoresis data, which are indicative of the genotype of the secondvariant site of the first sample polynucleotide. The genotype of thesecond variant site of the sample polynucleotide may be determined basedon the third and fourth electrophoresis data.

[0012] At least one and preferably both of the first and second duplexesmay have a size that differs from at least one and preferably both ofthe third and fourth duplexes. Subjecting the first and second duplexesto TGE and subjecting the third and fourth duplexes to TGE comprisesimultaneously subjecting at least 3 and preferably 4 duplexes of thefirst, second, third, and fourth duplexes to TGE along the sameseparation lane. At least one and preferably both of the first andsecond duplexes may have a size that differs from at least one of thethird and fourth duplexes by at least 20 base pairs.

[0013] The method may comprise amplifying at least a first portion of asecond different sample polynucleotide to obtain second amplicons. Thesecond sample polynucleotide comprises a second variant site. Theposition of the second variant site along the second samplepolynucleotide may be known. The second amplicons may include the secondvariant site of the sample polynucleotide. The second amplicons andthird and fourth different polynucleotide controls may be combined. Thethird and fourth polynucleotide controls may differ by at least one basetherealong. The position of the at least one differing base maycorrespond to the second variant site of the second samplepolynucleotide.

[0014] A plurality of third duplexes may be prepared. Each of at leastsome of the third duplexes may comprise (i) a polynucleotide strand ofone of the second amplicons and (ii) a complementary polynucleotidestrand of the third polynucleotide control. A plurality of fourthduplexes may be prepared. Each of at least some of the fourth duplexesmay comprise (i) a polynucleotide strand of one of the second ampliconsand (ii) a complementary polynucleotide strand of the fourthpolynucleotide control. The third and fourth duplexes may be subjectedto temperature gradient electrophoresis (TGE) to obtain third and fourthelectrophoresis data, which are indicative of the genotype of the secondvariant site of the sample polynucleotide. The genotype of the secondvariant site of the second sample polynucleotide may be determined basedon the third and fourth electrophoresis data.

[0015] At least one or both of the first and second duplexes may have asize that differs from at least one or both of the third and fourthduplexes. Subjecting the first and second duplexes to TGE and subjectingthe third and fourth duplexes to TGE may comprise simultaneouslysubjecting at least 3 and preferably 4 duplexes of the first, second,third, and fourth duplexes to TGE along the same separation lane. Atleast one of the first and second duplexes has a size that differs fromat least one of the third and fourth duplexes by at least 20 base pairs.

[0016] Another embodiment of the invention relates to a method fordetermining the genotype of a sample polynucleotide. The method maycomprise providing first and second polynucleotide controls. The firstand second polynucleotide controls may differ by at least one basetherealong. The position of the differing base may correspond to aposition of a variant site of the sample polynucleotide. The position ofthe variant site along the sample polynucleotide is preferably known.

[0017] A first amount of the sample polynucleotide may be combined withthe first polynucleotide control to prepare a first mixture. Each of thesample polynucleotide and the first polynucleotide control may comprisea polynucleotide strand sufficiently complementary to form a duplex witha polynucleotide strand of the other of the sample polynucleotide andfirst polynucleotide control.

[0018] First duplexes may be prepared. At least some of the firstduplexes may comprise a strand of the sample polynucleotide and a strandof the first polynucleotide control

[0019] A first amount of the sample polynucleotide with the secondpolynucleotide control to prepare a second mixture. Each of the samplepolynucleotide and the second polynucleotide control may comprise apolynucleotide strand sufficiently complementary to form a duplex with apolynucleotide strand of the other of the sample polynucleotide andsecond polynucleotide control.

[0020] Second duplexes may be prepared. At least some of the secondduplexes may comprise a strand of the sample polynucleotide and a strandof the second polynucleotide control.

[0021] The first and second mixtures may be subjected to temperaturegradient electrophoresis to obtain first and second electrophoresisdata, which is indicative of the genotype of the variant site of thesample polynucleotide. The genotype of the sample polynucleotide may bedetermined based on the first and second electrophoresis data.

[0022] Determining the genotype of the sample polynucleotide maycomprise determining a number of peaks present in the firstelectrophoresis data and a number of peaks present in the secondelectrophoresis data. One or both the first and second polynucleotidecontrols may be homozygous.

[0023] The sample polynucleotide may comprise one or more ampliconsprepared by amplifying at least one and preferably two first doublestranded polynucleotides. Each of the at least at least one first doublestranded polynucleotides may comprise genomic DNA of an organism such asmammal (e.g., a human) or a plant.

[0024] Another embodiment of the invention relates to a method fordetermining the genotype of a first variant site of a first samplepolynucleotide. The method may comprise providing amplicons of the ofthe first sample polynucleotide, the amplicons including the firstvariant site. A first portion of the amplicons may be subjected todenaturing and annealing to prepare a first mixture.

[0025] A first polynucleotide control may be provided. The firstpolynucleotide control may comprise at least one polynucleotide strandable to form a duplex with a polynucleotide strand of at least one ofthe amplicons. The first polynucleotide control may have a basecorresponding to the first variant site of the sample polynucleotide,the identity of the base may be known. The position of the variant sitealong the first sample polynucleotide may be known.

[0026] A second mixture may be prepared by combining a second portion ofthe amplicons with the first polynucleotide control. The second mixturemay be subjected to denaturing and annealing to prepare a third mixture.

[0027] The first mixture may be subjected to temperature gradientelectrophoresis (TGE) to obtain first electrophoresis data, which isindicative of the genotype of the first variant site. The second mixtureto temperature gradient electrophoresis (TGE) to obtain secondelectrophoresis data, which is indicative of the genotype of the firstvariant site. The method may comprise determining the genotype of thefirst variant site of the sample polynucleotide based on the first andsecond electrophoresis data.

[0028] The step of subjecting a first portion of the amplicons todenaturing and annealing to prepare a first mixture may be performedprior to introducing the amplicons to an electrophoresis separationlane, such as prior to injecting the first portion of amplicons into acapillary. The step of subjecting the second mixture to denaturing andannealing to prepare a third mixture may be performed prior tointroducing the second mixture to an electrophoresis separation lane,such as prior to injecting the second portion of amplicons into acapillary.

[0029] In one embodiment, the sample polynucleotide comprises a secondvariant site. The position of the second variant site along the samplepolynucleotide may be known. Second amplicons of the of the first samplepolynucleotide may be provided. The second amplicons may include thesecond variant site. A first portion of the second amplicons may besubjected to denaturing and annealing to prepare a fourth mixture.

[0030] A second polynucleotide control may be provided. The secondpolynucleotide control may comprise at least one polynucleotide strandable to form a duplex with a polynucleotide strand of at least one ofthe second amplicons. The second polynucleotide control may have a basecorresponding to the second variant site of the first samplepolynucleotide. The identity of the base may be known.

[0031] A second portion of the second amplicons may be combined with thesecond polynucleotide control to prepare a fifth mixture. The fifthmixture may be subjected to denaturing and annealing to prepare a sixthmixture.

[0032] The fourth mixture may be subjected to temperature gradientelectrophoresis (TGE) to obtain third electrophoresis data, which isindicative of the genotype of the second variant site.

[0033] The sixth mixture may be subjected to temperature gradientelectrophoresis (TGE) to obtain fourth electrophoresis data, which isindicative of the genotype of the second variant site. The genotype ofthe first variant site of the sample polynucleotide may be determinedbased on the first and second electrophoresis data.

[0034] The step of subjecting a first portion of the amplicons todenaturing and annealing to prepare a first mixture may be performedprior to introducing the amplicons to an electrophoresis separationlane, such as prior to injecting the first portion of amplicons into acapillary. The step of subjecting the second mixture to denaturing andannealing to prepare a third mixture may be performed prior tointroducing the second mixture to an electrophoresis separation lane,such as prior to injecting the second portion of amplicons into acapillary.

[0035] In one embodiment, the method comprises providing secondamplicons of a second sample polynucleotide. The second amplicons mayinclude a second variant site of the second sample polynucleotide. Theposition of the second variant site along the second samplepolynucleotide may be known.

[0036] A first portion of the second amplicons may be subjected todenaturing and annealing to prepare a fourth mixture.

[0037] A second polynucleotide control may be provided. The secondpolynucleotide control may comprise at least one polynucleotide strandable to form a duplex with a polynucleotide strand of at least one ofthe second amplicons. The second polynucleotide control may have a basecorresponding to the second variant site of the second samplepolynucleotide. The identity of the base may be known.

[0038] A second portion of the second amplicons may be combined with thesecond polynucleotide control to prepare a fifth mixture. The fifthmixture may be subjected to denaturing and annealing to prepare a sixthmixture.

[0039] The fourth mixture may be subjected to temperature gradientelectrophoresis (TGE) to obtain third electrophoresis data, indicativeof the second variant site of the second sample polynucleotide.

[0040] The sixth mixture to temperature gradient electrophoresis (TGE)to obtain fourth electrophoresis data, which is indicative of the secondvariant site of the second sample polynucleotide. The genotype of thesecond variant site of the second sample polynucleotide based on thefirst and second electrophoresis data.

[0041] The step of subjecting a first portion of the amplicons todenaturing and annealing to prepare a first mixture may be performedprior to introducing the amplicons to an electrophoresis separationlane, such as prior to injecting the first portion of amplicons into acapillary. The step of subjecting the fifth mixture to denaturing andannealing to prepare a third mixture may be performed prior tointroducing the second mixture to an electrophoresis separation lane,such as prior to injecting the second portion of amplicons into acapillary.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The present invention is discussed herein in reference to thedrawings in which:

[0043]FIG. 1 illustrates preparation of homoduplexes and heteroduplexes;

[0044]FIG. 2 illustrates temperature gradient electrophoresis separationof four species of homo- and heteroduplexes along a first separationlane in a sieving matrix and migration of a wild-type control along asecond separation lane in a sieving matrix;

[0045]FIG. 3 illustrates schematic electrophoresis data as may beobtained for different genotypes in determination of DNA variants withTGE using a homozygous control;

[0046]FIG. 4 shows electrophoresis data obtained in accordance with thescheme of FIG. 3: first genotype scores without addition of control tothe sample polynucleotides, two homozygous controls (CC and TT), Het:one heterozygous control;

[0047]FIG. 5: shows experimental data obtained with in accordance withthe scheme of FIG. 3, Second genotype scores with addition of a controlpolynucleotide to the sample polynucleotides;

[0048]FIG. 6 illustrates schematic electrophoresis data as may beobtained for different genotypes in determining DNA variants with TGEusing two homozygous controls;

[0049]FIG. 7 shows electrophoresis data obtained in accordance with thescheme of FIG. 6: first genotype scores with addition of a firsthomozygous control to the samples;

[0050]FIG. 8 shows electrophoresis data obtained in accordance with thescheme of FIG. 6: second genotype scores with addition of a secondhomozygous control to the samples;

[0051]FIG. 9 shows electrophoresis data obtained from a multiplexed runof eight heterozygous samples along a single separation lane, X-axis:Frame number (migration time), Y-axis: electrophoresis data intensity,peak numbers, corresponding DNA sizes and size difference in base pairsto next peak, PEO sieving matrix;

[0052]FIG. 10 shows detail of first four electrophoresis peaks of FIG.9;

[0053]FIG. 11 shows detail of last four electrophoresis peaks of FIG. 9;

[0054]FIG. 12 shows electrophoresis data obtained from a multiplexedseparation of three heterozygous samples along a single separation lane,X-axis: Frame number (migration time), Y-axis: signal intensity, peaknumbers, corresponding DNA sizes and size difference in base pairs tonext peak, PEO sieving matrix;

[0055]FIG. 13 shows electrophoresis data obtained from a multiplexedseparation of three homozygous controls, corresponding to the threeheterozygous samples of FIG. 12, X-axis: Frame number (migration time),Y-axis: signal intensity, peak numbers, corresponding DNA sizes and sizedifference in base pairs to next peak, PEO sieving matrix;

[0056]FIG. 14 shows a flowchart of a method of the invention;

[0057]FIG. 15 illustrates a user interface of the invention;

[0058]FIG. 16 illustrates use of two scores to make a genotype call fora sample polynucleotide;

[0059]FIG. 17 illustrates use of a panel of controls and/or molecularladders to define a migration zone for any specific sample;

[0060]FIG. 18 illustrates a decision table for using electrophoresisdata to determine whether a portion of a polynucleotide comprises avariant site, such as a polymorphism;

[0061]FIG. 19 illustrates a decision table for determining the genotypeof a variant site of a sample polynucleotide based on first and secondelectrophoresis data;

[0062]FIG. 20 illustrates schematic electrophoresis data as may beobtained by subjecting each of three different samples to TGE along thesame separation lane; and

[0063]FIG. 21 illustrates schematic electrophoresis data as may beobtained by subjecting different sized amplicons of a common portion ofa sample polynucleotide to TGE in the presence of different controlpolynucleotides along the same separation lane.

DETAILED DESCRIPTION OF THE INVENTION

[0064] The present invention provides a powerful tool for studies ofdisease associations using SNPs as the DNA markers. It may be used inpharmacgenomics to relate individual genotypes to drug usages. Theinvention may also be used for disease diagnostics. There is anincreasing demand for the genotyping technology for more efficientdetection of DNA variations. A technical system disclosed here issuitable to conduct high throughput analysis for known mutations andsingle nucleotide polymorphisms (SNPs). Unlike use of temperaturegradient electrophoresis for discovering unknown mutations (e.g., Gaoand Yueng, 1999), the method of the present invention may be used forgenotyping of known DNA variants (such as SNPs or mutations) in agenome. Thus, the exact locations of SNPs/mutations are determined inthe art. The method in accordance with Gao and Yueng requires asequencing step to locate the exact position of the mutation. In oneembodiment of the present invention, at least one and preferably two ormore homozygous DNA controls are added testing material in order toscore all three possible genotypes in a diploid organism. The testingmaterial may be analyzed in multiplexed fashion to increase efficiencyof the technique.

[0065] Referring to FIG. 1, a TGE assay in accordance with the inventionmay be based on a heteroduplex analysis in which a heterozygous samplewith a mutation/SNP is denatured and slowly annealed to formhomoduplexes of original strands of polynucleotide and heteroduplexeseach with a mismatch at the mutation/SNP site. In accordance with anyembodiment of the invention, excess primers, if present, may be removedprior to the denaturing and annealing steps. These four species of DNAmolecules have different melting temperatures (Tm), which are thetemperatures at which half of the double-stranded DNA moleculesdissociate (denature) and become single-stranded.

[0066] Referring to FIG. 2, a sample comprising duplexes is separated ina sieving medium preferably premixed with an intercalating dye. Themigrating duplexes are subjected to a temperature gradient. Even thoughthe homoduplexes having similar Tm's and the heteroduplexes have similarTm's, the Tms for two heteroduplexes are usually lower. Thus, the Tms oftwo heteroduplexes will be reached first. When the Tm is reached, duplexwill partially denature to form a bulky structure, which retards themigration of the duplex. The overall result is that the homoduplexes andheteroduplexes will separate from each other (FIG. 2). When, forexample, they pass through the detection window, electrophoresis datamay be obtained. The electrophoresis data is indicative of the separatedduplexes. For example, when heteroduplexes are present, theelectrophoresis data may contain more or broader peaks than will awild-type control subjected to temperature gradient electrophoresis.

[0067] Depending on the steepness of the temperate gradient, aheterozygous sample can be resolved into four, three, two peaks or evena broad peak, compared to the control. The closer the rampingtemperature to the Tm's of the sample, as well as the slower the rate ofthe ramp, the better the resolution can be achieved, i.e. more three-,four-peak patterns can be observed. However, if the purpose is todistinguish the mutation/SNP peak pattern from that of the control, onecan use a broader ramp to scan various samples with different Tm's in asingle test. The tested sample with a peak pattern distinguishable fromthe control will be sequenced in order to locate the exact position ofthe variant site. So, It may be unnecessary, in many cases, to resolvetesting material into its highest resolution.

[0068] To score all three possible genotypes (i.e. CC, CT and TT) in adiploid organism, two TGE assays may be used to reveal these known sitesof DNA variants. Each assay generates a genotype score for the testingsample. Scores obtained from two assays may be combined to produce afinal call of genotype for the sample.

[0069] One embodiment of the invention comprises subjecting one or moresample polynucleotides (and/or one or more amplicons corresponding to atleast a portion of the sample polynucleotides) to TGE in the absence ofa control. Electrophoresis data is obtained from the TGE. Theelectrophoresis data may be used to determine a first score (shownschematically in the upper half of FIG. 3). The first score may bereferred to as an initial determination of the genotype of each samplepolynucleotide. Referring to FIG. 4, electrophoresis data and firstscores for each of six sample polynucleotides are shown.

[0070] A second score may be obtained by subjecting the one or moresample polynucleotide (and/or one or more amplicons corresponding to atleast a portion of the sample polynucleotides) to TGE in the presence ofa control, which may be a homozygous or heterozygous control as shownschematically in the lower half of FIG. 3 showing use of a homozygouscontrol. Electrophoresis data is obtained from the TGE separation. Thecontrol is preferably a duplex comprising first and second singlestranded polynucleotides each preferably able to form a duplex with oneor more strands of the one or more sample polynucleotides and oramplicons derived from the sample polynucleotides. For example, wherethe sample polynucleotide and or amplicons comprise two complementarypolynucleotide strands, the control polynucleotide may comprise a firstpolynucleotide strand able to form a duplex with a first strand of thesample polynucleotide or amplicon and a second polynucleotide strandable to form a duplex with the complementary strand of the samplepolynucleotide or amplicon.

[0071] The control and the one or more sample polynucleotides and oramplicons corresponding thereto are preferably subjected to at least onedenaturing and annealing step prior to prepare duplexes. Referring toFIG. 5, electrophoresis data obtained from TGE of a TT control and ofthe sample polynucleotides of FIG. 4. The duplexes of the control andsample polynucleotides are prepared prior to obtaining theelectrophoresis data and preferably prior to migrating the control andsample polynucleotides along an electrophoresis separation lane. Secondscores obtained from the electrophoresis data FIG. 4 are also shown asare final scores indicative of the genotype of each of the six samplepolynucleotides.

[0072] Referring to FIG. 6, another embodiment of the inventioncomprises subjecting the sample polynucleotide (and/or one or moreamplicons corresponding to at least a portion of the samplepolynucleotides) to TGE in the presence of at least two controls, whichmay be, for example, homozygous or heterozygous. Electrophoresis data isobtained from the TGE separation. The electrophoresis data may be usedto determine first and second scores for the sample polynucleotides oramplicons corresponding thereto. FIGS. 6, 7 and 8 schematicallyillustrates use of homozygous CC and TT controls.

[0073] Although homozygous controls are preferred, heterozygous controlsmay be used. Each control is preferably a duplex comprising first andsecond single stranded polynucleotides each preferably able to form aduplex with a single strand of the sample polynucleotide and oramplicons derived from the sample polynucleotide. For example, where thesample polynucleotide and or amplicons comprise two complementarypolynucleotide strands, the control polynucleotide may comprise a firstpolynucleotide strand able to form a duplex with a first strand of thesample polynucleotide or amplicon and a second polynucleotide strandable to form a duplex with the complementary strand of the samplepolynucleotide or amplicon.

[0074] Preferably, the sample polynucleotide is subjected to TGE in thepresence of one of the controls along a first separation lane (upperhalf of FIG. 6). Along a second separation lane, or along the firstseparation lane at a preferably different time, the samplepolynucleotide is subjected to TGE in the presence of a differentcontrol, which may be of the same type or different as the first control(lower half of FIG. 6). As shown schematically in FIG. 6, theelectrophoresis data obtained from the TGE depends upon the control usedand the genotype of the sample polynucleotide being tested. FIG. 7 showselectrophoresis data obtained upon subjecting a sample polynucleotide toTGE in the presence of a CC control polynucleotide following duplexformation. FIG. 8 shows electrophoresis data obtained upon subjecting apolynucleotide to TGE in the presence of a TT control polynucleotidefollowing duplex formation. Second scores and final scores obtained fromthe electrophoresis data FIGS. 7 and 8 are also shown in FIG. 8. Ineither strategy, multiplexed samples with different lengths of DNAamplicons can be separated in along a single separation lane to achievemaximum efficiency of the technique (FIGS. 9, 10, 11, 12, and 13).

[0075] Referring to the flow chart of FIG. 14, in one embodiment, themethod of the invention comprises amplification, such as by polymerasechain reaction (PCR), of regions of interest of a polynucleotidecontaining a mutation or a SNP to prepare amplicons (FIG. 14). In anyembodiment of the invention, the polynucleotide may be DNA, such asgenomic DNA of an organism. The organism may be, for example, an animal(including mammals) or a plant. In a preferred embodiment, thepolynucleotide is of a human.

[0076] The amplification may be performed by one or more amplificationreactions, each of which amplifies a single polynucleotide region ofinterest or by a multiplexed reaction, which concomitantly amplifies aplurality of preferably different regions of interest. Differentamplified regions of the polynucleotide may overlap, i.e., may includecommon regions of the polynucleotide. In any event, the amplificationreaction provides amplicons, which may be PCR products. Amplicons of themay be, for example, wild-type homozygote, mutation homozygote, mutationheterozygote, or combinations thereof.

[0077] Amplicons prepared in accordance of the invention may be ofdifferent sizes. For example, the amplicons may have sizes of about 20base pairs to about 1000 or even more base pairs. Amplicons of differentsizes may have sequences that correspond to different regions of thepolynucleotide. Alternatively, or in combination, amplicons of differentsizes may be prepared from different polynucleotides. A plurality of theamplicons may be combined or pooled. For example, 5, 15, 20 or moreamplicons can be pooled from individual and/or multiplex PCR reactionswithout any further post-PCR purification. As a consideration, amultiplexed PCR reaction or samples to be pooled and separated in asingle channel with TGE preferably contain unrelated DNA sequences toprevent cross pairing of DNA strands from different amplicons. One ormore duplexes, such as heteroduplexes and/or homoduplexes, may beprepared from the amplicons with or without pooling of amplicons ofdifferent sizes. For example, duplexes may be prepared by subjecting oneor more amplicons to a denaturing and annealing step. Suitable methodsfor preparation of duplexes are disclosed in Applicant's copending U.S.application Ser. No. 10/287,826, filed Nov. 5, 2002, and incorporatedherein.

[0078] The duplexes are subjected to a temperature gradient separation,such as (TGE), along one or more separation lanes. The separation lanesmay be, for example, electrophoresis lanes such as capillaries, slabgels, or microfluidic structures. During electrophoresis, the migratingduplexes are subjected to a temperature ramp spanning meltingtemperatures (Tm's) for the homoduplexes and heteroduplexes.Heteroduplexes generally have lower Tm's than their correspondinghomoduplexes due to the presence of a mismatch. Thus, heteroduplexesdenature at a lower temperature than homoduplexes and will exhibit aretarded mobility in the gel, resulting in separation of heteroduplexand homoduplex species having the same lengths. Suitable temperaturegradient electrophoresis methods and systems are disclosed in thecopending 10/287,826 application.

[0079] Electrophoresis data indicative of the presence of the migratingheteroduplexes and homoduplexes is obtained, such as by using alaser-induced fluorescence detection system. The electrophoresis datamay be used to distinguish amplicons indicative of the presence of (i) amutation and or SNP from (ii) wild type.

[0080] One embodiment of the present invention relates to a computerreadable medium comprising code. The code may prepare data, such as avisual display or printout, identifying the presence of DNA variants andpreferably their genotypes. The data may be prepared automatically usingelectrophoresis data obtained from a TGE separation. Preferably, thecomputer readable medium comprises code to call a genotype for a testsample with each addition of two controls, and then combined two callsto generate a final call for a genotype of the testing sample; 2)defining a migration zone for a specific PCR product for amultiplexed-sample separation by TGE using a molecular ladder and/or aDNA control panel as a size calibration. The control panel can beassembled based on multiplexed samples, which are electrophoresed in aseparate channel.

[0081] Sample preparation.

[0082] To take full advantage of the separation capability of anyinstrumental platform (such as an automated capillary sequencinginstrument), primer pairs for amplifying different DNA regions can besuch designed that PCR products ranging from 100 to 800 bp will begenerated. To ensure a clear separation of different lengths of DNAfragments, each PCR amplicon should be generated with 30-50 bp apart.Each PCR product can be a wild-type homozygote, a mutation homozygote ora mutation heterozygote. The PCR reaction can be performed with a singlepair of primers for a single amplicon or multiple pairs of primers formultiple amplicons. The latter strategy would further reduce the time,labor and the cost of reagents for the assay. Multiplex PCR reactions,as disclosed in Elnifro et al. 2000, are known to those in the art.

[0083] Multiplexing efficiency depends on how many base pairs separatethe lengths of neighboring DNA fragments. The fewer the base pairs, thegreater the multiplexing efficiency. FIG. 10 shows temperatureelectrophoresis data obtained for polynucleotides differing in size byfrom 20 to 119 base pairs. Therefore, one can easily multiplex 20samples in a lane that can separated DNA fragments up to 800 bp.Moreover, the electrophoresis data of FIG. 9 demonstrate that a singletemperature ramp, in this case 8° C., may be used to separate aplurality of samples present in a mixture. Therefore, the invention willprovide not only a high-throughput method, but also a cost-efficientmeans to conduct DNA genotyping. For example, approximately 1,000samples may be analyzed in two hours in a single run using a96-capillary electrophoresis device, if 20 samples/capillary areincluded, with a cost of 1-2 cents. An example of a suitableelectrophoresis device is disclosed in U.S. patent application Ser. No.10/287,826, filed Nov. 5, 2002, which is incorporated herein in itsentirety.

[0084] Addition of homozygous controls to the testing samples andmultiplexed analysis.

[0085] Control DNA amplicons can be prepared in large quantity and addto the testing samples. The amplification efficiency for an ampliconwith a same pair of primers targeting a same region of DNA is usuallysimilar. Thus, a 1:1 mixture of the control and testing samples isusually sufficient for any amplicons. Even if there are smalldifferences on concentrations of some amplicons between the control andthe sample, it will not affect the final call since the peak patternwill still be very similar. We have resolved samples with 1:40difference in concentration.

[0086] TGE separation.

[0087] The homo- and heteroduplexes formed through denaturing andannealing process are then separated by the TGE method and detected bythe LIF system. Using the TGCE system developed by SpectruMedixCorporation, the temperature gradient is provided by bathing capillaryarray in hot air that is circulated through a heater that is externallycontrolled by the instrument control computer. For TGCE analysis, crudesamples can be directly injected into capillaries. The polyethyleneoxide (PEO) gel matrix is used for electrophoresis. Optimization of thetemperature profile for each sample is not required since the selectedtemperature ramp will cover Tms for all samples tested in the run. Thetemperature controller performs a predetermined temperature ramp,typically at a rate of 0.4 C/min. Under these conditions, theheteroduplex reaches the Tm earlier than its corresponding homoduplexdue to the mismatch and thus exhibit a retarded mobility in the gel,resulting in separation from the homoduplex. Fluorescence from anintercalating dye is excited with an air-cooled argon ion laser at allline emission mode. A CCD camera was used to detect fluorescence fromall 96 capillaries simultaneously. SpectruMedix CheckMate® software maybe used for instrument control and data acquisition.

[0088] Automated software to report genotypes of DNA variants.

[0089] A computer program is created to report genotypes of DNA variantsautomatically after the TGE separation. There are two key features ofthe software: 1) calling a genotype for a test sample with each additionof two controls, and then combined two calls to generate a final callfor a genotype of the testing sample; 2) defining a migration zone for aspecific PCR product for a multiplexed-sample separation by TGE using amolecular ladder and/or a DNA control panel as a size calibration. Thecontrol panel can be assembled based on multiplexed samples, which areelectrophoresed in a separate channel.

[0090] In practice, one may divide a 24-, 48-, 96-, or 384-well trayinto two halves. One half runs original testing DNA samples, the otherhalf runs the same set of testing samples mixed with one of thecontrols. Alternatively, one half runs the testing samples mixed withone control and the other half runs the same set of testing samplesmixed with the other control. FIG. 15 shows a program interface on howthe controls and a pair of the same set of sample can be selected andcompared. The middle panel is the layout of a 96-well tray on which sixsuch samples were run to prove the principle of concept. Samples 1-6were mixed with the control CC and added to the wells 37-42. Whereaswells 49-54 were used to hold the same set of samples mixed with theother control TT. Wells 43, 44 and 45 contained CC, CT and TT controls.

[0091] One may select CC or TT polynucleotides (or combination thereof)as the control for samples in wells 37-42 or wells 49-54 to compare thepeak patterns to see if any DNA variant is present. One then matches thewells of same sample for the down stream report. The up-panel of FIG. 16shows the individual calls for samples mixed with either of thecontrols. The lower-panel indicates the final genotype scores for eachsample. FIG. 17 shows an example of using a panel of controls asmolecular ladders to define a migration zone for a specific PCR productin a multiplexed-sample separation by TGE.

[0092] As shown in FIG. 18, one method of the invention comprises use ofTGCE as a screening process. The data indicate the presence or absenceof a polymorphism. Once a polymorphism is identified, “positive hits”are screened by conventional means. The benefit with a TGCE screeningprocess is the reduction of the need to sequence large areas where thereare no polymorphisms. In accordance with the method, a homoduplexstandard may be subjected to TGCE in a separate lane. A determination ofthe variant state of the polynucleotide is based on comparing theunknown migration profile with the standard. A preferred embodiment ofthe current process employs an intercalating fluorescent dye (e.g.,ethidium bromide). This reduces the cost of the analysis and simplifiesthe sample preparation. In this embodiment the analysis may employsingle color detection.

[0093] As compared to known approaches, the present invention increasesthe amount of information obtained. The specific identity of thepolymorphism may also be determined. Another distinction between thecurrent approach and the present invention is the absence of arequirement that any homoduplex standard be run in another separationlane. The data obtained from running a matrix, such as a 2×3, ofpossibilities eliminates the need for running pure samples. Anotherdistinction is that multiple duplex pairs differing in size may becombined and simultaneously subjected to TGE. This provides the abilityto multiplex the number of mutation samples per electrophoretic lane.The end result of such multiplexing is a panel of duplexes, separated intime in the electrophoresis due to differing fragment size.

[0094] One embodiment of the present invention relates to a method fordetermining the identity of first and second bases of a DNA compound.First and second DNA control compounds are prepared. The first andsecond control DNA compounds differ by at least one base therealong. Forexample, one control compound may be CC and the other compound may be TTwith respect to a particular site within the control compound. The firstand second control DNA compounds may be duplexes.

[0095] The DNA compound may be combined with the first and secondcontrol DNA to form first and second mixtures. The mixtures may besubjected to at least one heating and cooling cycle to formheteroduplexes. The first and second mixtures are subjected totemperature gradient electrophoresis. The identity of the first andsecond bases of the DNA compound (i.e. the genotype of the DNA compound)is determined based on peaks obtained in the temperature gradientelectrophoresis.

[0096] Referring to FIG. 19, for example, the row designated CC showsthe peak pattern expected if the DNA compound is of the CC genotype.Temperature gradient electrophoresis (TGE) in the presence of the CCcontrol compound produces a single or narrow peak while TGE in thepresence of the TT control compound produces a plurality of peaks or asingle wider peak. In general, the CC genotype is determined if TGEproduces a wider peak or plurality of peaks in the presence of the TTcontrol compound than in the presence of the CC control compound.Similarly, referring to row 3, the presence of the TT genotype isdetermined if TGE produces a wider peak or plurality of peaks in thepresence of the CC control compound than in the presence of the TTcontrol compound. Referring to row 2 of FIG. 19, the CT genotype isdetermined if both TGE channels produce a plurality of peaks or a widerpeak than expected for a single compound.

[0097] Peaks can be compared on the basis of, for example, peak width,such as full width half maximum, and peak area. The number of peaks canbe determined by, for example, a derivative filter, such as a SavitzkyGolay filter. The peaks obtained in a given TGE run can be compared topeaks in a look-up table to determine whether one or more peaks arepresent.

[0098]FIG. 19 may be summarized according to the following Booleantable:

[0099] If [1 peak] is observed with CC, and [plurality or wider peakswith TT; then sample polynucleotide=CC

[0100] If [plurality of peaks] with CC, and [plurality of peaks] withTT; then sample polynucleotide=CT

[0101] If [plurality of peaks or wider] with CC, and [1 peak] with TT;then sample polynucleotide=TT.

[0102] Referring to FIGS. 18 and 19, the method may be multiplexed toallow more than one mixture to be simultaneously subjected to TGE withina single separation lane. Each sample has a different migration time.Samples, having different sizes, are pooled together and subjected toTGE. Peaks within a given migration time range are analyzed to determinethe presence of single or multiple peaks, as discussed above.

[0103] Referring to FIGS. 20 and 21, any method of the invention may bemultiplexed to allow more than one mixture to be simultaneouslysubjected to TGE within a single separation lane. Each sample preferablya different migration time. Samples, having different sizes, are pooledtogether and subjected to TGE. Electrophoresis data is obtained. Thedata is indicative of the genotype of the sample polynucleotide orsample polynucleotides. Peaks within a given migration time range may beanalyzed to determine the presence of single or multiple peaks, asdiscussed above.

[0104] The following references are incorporated to the extent necessaryto understand the present invention:

[0105] A supplement to BioTechniques, SNPs: Discovery of markers fordisease. June 2002.

[0106] Applied Biosystems, 2000, Protocol of ABI Prism SNaPshotMultiplex Kit.

[0107] Elnifro E., Ashshi A., Cooper R., and Klapper P. 2000. ClinicalMicrobio. Rev. 13:559-570.

[0108] Igloi G. 2001. Genomics 74:402-407.

[0109] Landergren U. M., Nilsson, and P-Y. Kwok. 1998. Genome Res.8:767-776.

[0110] Ray R. and Norden B. 2000. The FASEB J. 14:1041-1060.

[0111] Ross P., Hall L., Smirnov I., and Haff, L. 1998. NatureBiotechnol. 16:1347-1351.

[0112] Wang D. G. et al., 1998. Science. 280:1077-1082.

What is claimed is:
 1. A method of determining the genotype of a samplepolynucleotide having at least a first variant site, comprising:amplifying at least a portion of the sample polynucleotide to obtainfirst amplicons, the first amplicons including the first variant site;combining the first amplicons with first and second differentpolynucleotide controls, the first and second polynucleotide controlsdiffering by at least one base therealong, the position of the at leastone differing base corresponding to the first variant site of the samplepolynucleotide; preparing a plurality of first duplexes, each of atleast some of the first duplexes comprising (i) a polynucleotide strandof one of the first amplicons and (ii) a complementary polynucleotidestrand of the first polynucleotide control; preparing a plurality ofsecond duplexes, each of at least some of the second duplexes comprising(i) a polynucleotide strand of one of the first amplicons and (ii) acomplementary polynucleotide strand of the second polynucleotidecontrol; subjecting the first and second duplexes to temperaturegradient electrophoresis (TGE) to obtain first and secondelectrophoresis data; and determining the genotype of the first variantsite of the sample polynucleotide based on the first and secondelectrophoresis data.
 2. The method of claim 1, wherein determining thegenotype of the sample polynucleotide comprises determining a number ofpeaks present in the first electrophoresis data and a number of peakspresent in the second electrophoresis data.
 3. The method of claim 1,wherein the first duplexes and second duplexes are subjected to TGEalong first and second different separation lanes.
 4. The method ofclaim 1, wherein the first and second polynucleotide controls arewild-type polynucleotides.
 5. The method of claim 1, comprising:amplifying at least a second different portion of the samplepolynucleotide to obtain second amplicons, the second ampliconsincluding a second variant site of the sample polynucleotide; combiningthe second amplicons with third and fourth different polynucleotidecontrols, the third and fourth polynucleotide controls differing by atleast one base therealong, the position of the at least one differingbase corresponding to the second variant site of the samplepolynucleotide; preparing a plurality of third duplexes, each of atleast some of the third duplexes comprising (i) a polynucleotide strandof one of the second amplicons and (ii) a complementary polynucleotidestrand of the third polynucleotide control; preparing a plurality offourth duplexes, each of at least some of the fourth duplexes comprising(i) a polynucleotide strand of one of the second amplicons and (ii) acomplementary polynucleotide strand of the fourth polynucleotidecontrol; subjecting the third and fourth duplexes to temperaturegradient electrophoresis (TGE) to obtain third and fourthelectrophoresis data; and determining the genotype of the second variantsite of the sample polynucleotide based on the third and fourthelectrophoresis data.
 6. The method of claim 5, wherein at least one ofthe first and second duplexes has a size that differs from at least oneof the third and fourth duplexes and wherein subjecting the first andsecond duplexes to TGE and subjecting the third and fourth duplexes toTGE comprise simultaneously subjecting at least 3 duplexes of the first,second, third, and fourth duplexes to TGE along the same separationlane.
 7. The method of claim 6, wherein at least one of the first andsecond duplexes has a size that differs from at least one of the thirdand fourth duplexes by at least 20 base pairs.
 8. The method of claim 1,comprising: amplifying at least a first portion of a second differentsample polynucleotide to obtain second amplicons, the second samplepolynucleotide comprising a second variant site, the second ampliconsincluding the second variant site of the sample polynucleotide;combining the second amplicons with third and fourth differentpolynucleotide controls, the third and fourth polynucleotide controlsdiffering by at least one base therealong, the position of the at leastone differing base corresponding to the second variant site of thesecond sample polynucleotide; preparing a plurality of third duplexes,each of at least some of the third duplexes comprising (i) apolynucleotide strand of one of the second amplicons and (ii) acomplementary polynucleotide strand of the third polynucleotide control;preparing a plurality of fourth duplexes, each of at least some of thefourth duplexes comprising (i) a polynucleotide strand of one of thesecond amplicons and (ii) a complementary polynucleotide strand of thefourth polynucleotide control subjecting the third and fourth duplexesto temperature gradient electrophoresis (TGE) to obtain third and fourthelectrophoresis data; and determining the genotype of the second variantsite of the sample polynucleotide based on the third and fourthelectrophoresis data.
 9. The method of claim 8, wherein at least one ofthe first and second duplexes has a size that differs from at least oneof the third and fourth duplexes and wherein subjecting the first andsecond duplexes to TGE and subjecting the third and fourth duplexes toTGE comprise simultaneously subjecting at least 3 duplexes of the first,second, third, and fourth duplexes to TGE along the same separationlane.
 10. The method of claim 9, wherein at least one of the first andsecond duplexes has a size that differs from at least one of the thirdand fourth duplexes by at least 20 base pairs.
 11. A method fordetermining the genotype of a sample polynucleotide, comprising:providing first and second polynucleotide controls, the first and secondpolynucleotide controls differing by at least one base therealong, theposition of the differing base corresponding to a position of a variantsite of the sample polynucleotide; combining a first amount of thesample polynucleotide with the first polynucleotide control to prepare afirst mixture, each of the sample polynucleotide and the firstpolynucleotide control comprising a polynucleotide strand sufficientlycomplementary to form a duplex with a polynucleotide strand of the otherof the sample polynucleotide and first polynucleotide control; formingfirst duplexes, at least some of the first duplexes comprising a strandof the sample polynucleotide and a strand of the first polynucleotidecontrol; combining a first amount of the sample polynucleotide with thesecond polynucleotide control to prepare a second mixture, each of thesample polynucleotide and the second polynucleotide control comprising apolynucleotide strand sufficiently complementary to form a duplex with apolynucleotide strand of the other of the sample polynucleotide andsecond polynucleotide control; subjecting the first and second mixturesto temperature gradient electrophoresis to obtain first and secondelectrophoresis data; and determining the genotype of the samplepolynucleotide based on the first and second electrophoresis data. 12.The method of claim 11, wherein determining the genotype of the samplepolynucleotide comprises determining a number of peaks present in thefirst electrophoresis data and a number of peaks present in the secondelectrophoresis data.
 13. The method of claim 11, wherein both the firstand second polynucleotide controls are homozygous.
 14. The method ofclaim 11, wherein the sample polynucleotide comprises an ampliconprepared by amplifying a first double stranded polynucleotide.
 15. Amethod for determining the genotype of a first variant site of a firstsample polynucleotide, comprising: providing amplicons of the of thefirst sample polynucleotide, the amplicons including the first variantsite; subjecting a first portion of the amplicons to denaturing andannealing to prepare a first mixture; providing a first polynucleotidecontrol, the first polynucleotide control comprising at least onepolynucleotide strand able to form a duplex with a polynucleotide strandof at least one of the amplicons, the first polynucleotide controlhaving a base corresponding to the first variant site of the samplepolynucleotide, the identity of the base being known; combining a secondportion of the amplicons with the first polynucleotide control toprepare a second mixture subjecting the second mixture to denaturing andannealing to prepare a third mixture; subjecting the first mixture totemperature gradient electrophoresis (TGE) to obtain firstelectrophoresis data; subjecting the second mixture to temperaturegradient electrophoresis (TGE) to obtain second electrophoresis data;and wherein the first and second electrophoresis data are indicative ofthe genotype of the first variant site of the first samplepolynucleotide.
 16. The method of claim 15, comprising determining thegenotype of the first variant site of the sample polynucleotide based onthe first and second electrophoresis data.
 17. The method of claim 15,wherein the step of subjecting a first portion of the amplicons todenaturing and annealing to prepare a first mixture is performed priorto introducing the amplicons to an electrophoresis separation lane. 18.The method of claim 15, wherein the step of subjecting the secondmixture to denaturing and annealing to prepare a third mixture isperformed prior to introducing the second mixture to an electrophoresisseparation lane.
 19. The method of claim 15, wherein the samplepolynucleotide comprises a second variant site and the method comprises:providing second amplicons of the of the first sample polynucleotide,the second amplicons including the second variant site; subjecting afirst portion of the second amplicons to denaturing and annealing toprepare a fourth mixture; providing a second polynucleotide control, thesecond polynucleotide control comprising at least one polynucleotidestrand able to form a duplex with a polynucleotide strand of at leastone of the second amplicons, the second polynucleotide control having abase corresponding to the second variant site of the first samplepolynucleotide, the identity of the base being known; combining a secondportion of the second amplicons with the second polynucleotide controlto prepare a fifth mixture subjecting the fifth mixture to denaturingand annealing to prepare a sixth mixture; subjecting the fourth mixtureto temperature gradient electrophoresis (TGE) to obtain thirdelectrophoresis data; subjecting the sixth mixture to temperaturegradient electrophoresis (TGE) to obtain fourth electrophoresis data;and wherein the third and fourth electrophoresis data are indicative ofthe genotype of the second variant site of the first samplepolynucleotide.
 20. The method of claim 19, comprising determining thegenotype of the first variant site of the sample polynucleotide based onthe first and second electrophoresis data.
 21. The method of claim 19,wherein the step of subjecting a first portion of the amplicons todenaturing and annealing to prepare a first mixture is performed priorto introducing the amplicons to an electrophoresis separation lane. 22.The method of claim 19, wherein the step of subjecting the secondmixture to denaturing and annealing to prepare a third mixture isperformed prior to introducing the second mixture to an electrophoresisseparation lane.
 23. The method of claim 15, comprising: providingsecond amplicons of a second sample polynucleotide, the second ampliconsincluding a second variant site of the second sample polynucleotide;subjecting a first portion of the second amplicons to denaturing andannealing to prepare a fourth mixture; providing a second polynucleotidecontrol, the second polynucleotide control comprising at least onepolynucleotide strand able to form a duplex with a polynucleotide strandof at least one of the second amplicons, the second polynucleotidecontrol having a base corresponding to the second variant site of thesecond sample polynucleotide, the identity of the base being known;combining a second portion of the second amplicons with the secondpolynucleotide control to prepare a fifth mixture subjecting the fifthmixture to denaturing and annealing to prepare a sixth mixture;subjecting the fourth mixture to temperature gradient electrophoresis(TGE) to obtain third electrophoresis data; subjecting the sixth mixtureto temperature gradient electrophoresis (TGE) to obtain fourthelectrophoresis data; and wherein the third and fourth electrophoresisdata are indicative of the genotype of the second variant site of thesecond sample polynucleotide.
 24. The method of claim 23, comprisingdetermining the genotype of the first variant site of the samplepolynucleotide based on the first and second electrophoresis data. 25.The method of claim 23, wherein the step of subjecting a first portionof the amplicons to denaturing and annealing to prepare a first mixtureis performed prior to introducing the amplicons to an electrophoresisseparation lane.
 26. The method of claim 23, wherein the step ofsubjecting the second mixture to denaturing and annealing to prepare athird mixture is performed prior to introducing the second mixture to anelectrophoresis separation lane.